Accelerated cardiomyocyte senescence contributes to late-onset doxorubicin-induced cardiotoxicity.

Children surviving cancer and chemotherapy are at risk for adverse health events including heart failure that may be delayed by years. Although the early effects of doxorubicin-induced cardiotoxicity may be attributed to a direct effect on the cardiomyocytes, the mechanisms underlying the delayed or late effects (8-20 yr) are unknown. The goal of this project was to develop a model of late-onset doxorubicin-induced cardiotoxicity to better delineate the underlying pathophysiology responsible. The underlying hypothesis was that doxorubicin-induced "late-onset cardiotoxicity" was the result of mitochondrial dysfunction leading to cell failure and death. Wistar rats, 3-4 wk of age, were randomly assigned to vehicle or doxorubicin injection groups (1-45 mg/kg). Cardiovascular function was unaltered at the lower dosages (1-15 kg/mg), but beginning at 6 mo after injection significant cardiac degradation was observed in the 45 mg/kg group. Doxorubicin significantly increased myocardial mitochondrial DNA (mtDNA) damage. In contrast, in isolated c-kit left ventricular (LV) cells, doxorubicin treatment did not increase mtDNA damage. Biomarkers of senescence within the LV were significantly increased, suggesting accelerated aging of the LV. Doxorubicin also significantly increased LV histamine content suggestive of mast cell activation. With the use of flow cytometry, a significant expansion of the c-kit and stage-specific embryonic antigen 1 cell populations within the LV were concomitant with significant decreases in the circulating peripheral blood population of these cells. These results are consistent with the concept that doxorubicin induced significant damage to the cardiomyocyte population and that although the heart attempted to compensate it eventually succumbed to an inability for self-repair.

Large-Scale Generation and Characterization of Homogeneous Populations of Migratory Cortical Interneurons from Human Pluripotent Stem Cells.

During development, cortical interneurons (cINs) are generated from the ventral telencephalon, robustly migrate to the dorsal telencephalon, make local synaptic connections, and critically regulate brain circuitry by inhibiting other neurons. Thus, their abnormality is associated with various brain disorders. Human pluripotent stem cell (hPSC)-derived cINs can provide unlimited sources with which to study the pathogenesis mechanism of these disorders as well as provide a platform to develop novel therapeutics. By employing spinner culture, we could obtain a >10-fold higher yield of cIN progenitors compared to conventional culture without affecting their phenotype. Generated cIN spheres can be maintained feeder-free up to 10 months and are optimized for passaging and cryopreservation. In addition, we identified a combination of chemicals that synchronously matures generated progenitors into SOX6+KI67- migratory cINs and extensively characterized their maturation in terms of metabolism, migration, arborization, and electrophysiology. When transplanted into mouse brains, chemically matured migratory cINs generated grafts that efficiently disperse and integrate into the host circuitry without uncontrolled growth, making them an optimal cell population for cell therapy. Efficient large-scale generation of homogeneous migratory cINs without the need of feeder cells will play a critical role in the full realization of hPSC-derived cINs for development of novel therapeutics.

Nuclear Gene 33/Mig6 regulates the DNA damage response through an ATM serine/threonine kinase-dependent mechanism.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently linked Gene 33 to the DNA damage response (DDR) induced by hexavalent chromium (Cr(VI)), but the molecular mechanism remains unknown. Here we show that ectopic expression of Gene 33 triggers DDR in an ATM serine/threonine kinase (ATM)-dependent fashion and through pathways dependent or not dependent on ABL proto-oncogene 1 non-receptor tyrosine kinase (c-Abl). We observed the clear presence of Gene 33 in the nucleus and chromatin fractions of the cell. We also found that the nuclear localization of Gene 33 is regulated by its 14-3-3-binding domain and that the chromatin localization of Gene 33 is partially dependent on its ErbB-binding domain. Our data further indicated that Gene 33 may regulate the targeting of c-Abl to chromatin. Moreover, we observed a clear association of Gene 33 with histone H2AX and that ectopic expression of Gene 33 promotes the interaction between ATM and histone H2AX without triggering DNA damage. In summary, our results reveal nuclear functions of Gene 33 that regulate DDR. The nuclear localization of Gene 33 also provides a spatial explanation of the previously reported regulation of apoptosis by Gene 33 via the c-Abl/p73 pathway. On the basis of these findings and our previous studies, we propose that Gene 33 is a proximal regulator of DDR that promotes DNA repair.

Inhibition of Histone Methyltransferase, Histone Deacetylase, and ß-Catenin Synergistically Enhance the Cardiac Potential of Bone Marrow Cells.

Previously, we reported that treatment with the G9a histone methyltransferase inhibitor BIX01294 causes bone marrow mesenchymal stem cells (MSCs) to exhibit a cardiocompetent phenotype, as indicated by the induction of the precardiac markers Mesp1 and brachyury. Here, we report that combining the histone deacetylase inhibitor trichostatin A (TSA) with BIX01294 synergistically enhances MSC cardiogenesis. Although TSA by itself had no effect on cardiac gene expression, coaddition of TSA to MSC cultures enhanced BIX01294-induced levels of Mesp1 and brachyury expression 5.6- and 7.2-fold. Moreover, MSCs exposed to the cardiogenic stimulus Wnt11 generated 2.6- to 5.6-fold higher levels of the cardiomyocyte markers GATA4, Nkx2.5, and myocardin when pretreated with TSA in addition to BIX01294. MSC cultures also showed a corresponding increase in the prevalence of sarcomeric protein-positive cells when treated with these small molecule inhibitors. These results correlated with data showing synergism between (1) TSA and BIX01294 in promoting acetylation of lysine 27 on histone H3 and (2) BIX01294 and Wnt11 in decreasing ß-catenin accumulation in MSCs. The implications of these findings are discussed in light of observations in the early embryo on the importance of ß-catenin signaling and histone modifications for cardiomyocyte differentiation and heart development.

Inhibition of G9a Histone Methyltransferase Converts Bone Marrow Mesenchymal Stem Cells to Cardiac Competent Progenitors.

The G9a histone methyltransferase inhibitor BIX01294 was examined for its ability to expand the cardiac capacity of bone marrow cells. Inhibition of G9a histone methyltransferase by gene specific knockdown or BIX01294 treatment was sufficient to induce expression of precardiac markers Mesp1 and brachyury in bone marrow cells. BIX01294 treatment also allowed bone marrow mesenchymal stem cells (MSCs) to express the cardiac transcription factors Nkx2.5, GATA4, and myocardin when subsequently exposed to the cardiogenic stimulating factor Wnt11. Incubation of BIX01294-treated MSCs with cardiac conditioned media provoked formation of phase bright cells that exhibited a morphology and molecular profile resembling similar cells that normally form from cultured atrial tissue. Subsequent aggregation and differentiation of BIX01294-induced, MSC-derived phase bright cells provoked their cardiomyogenesis. This latter outcome was indicated by their widespread expression of the primary sarcomeric proteins muscle α-actinin and titin. MSC-derived cultures that were not initially treated with BIX01294 exhibited neither a commensurate burst of phase bright cells nor stimulation of sarcomeric protein expression. Collectively, these data indicate that BIX01294 has utility as a pharmacological agent that could enhance the ability of an abundant and accessible stem cell population to regenerate new myocytes for cardiac repair.

The histone methyltransferase inhibitor BIX01294 enhances the cardiac potential of bone marrow cells.

Bone marrow (BM) has long been considered a potential stem cell source for cardiac repair due to its abundance and accessibility. Although previous investigations have generated cardiomyocytes from BM, yields have been low, and far less than produced from ES or induced pluripotent stem cells (iPSCs). Since differentiation of pluripotent cells is difficult to control, we investigated whether BM cardiac competency could be enhanced without making cells pluripotent. From screens of various molecules that have been shown to assist iPSC production or maintain the ES cell phenotype, we identified the G9a histone methyltransferase inhibitor BIX01294 as a potential reprogramming agent for converting BM cells to a cardiac-competent phenotype. BM cells exposed to BIX01294 displayed significantly elevated expression of brachyury, Mesp1, and islet1, which are genes associated with embryonic cardiac progenitors. In contrast, BIX01294 treatment minimally affected ectodermal, endodermal, and pluripotency gene expression by BM cells. Expression of cardiac-associated genes Nkx2.5, GATA4, Hand1, Hand2, Tbx5, myocardin, and titin was enhanced 114, 76, 276, 46, 635, 123, and 5-fold in response to the cardiogenic stimulator Wnt11 when BM cells were pretreated with BIX01294. Immunofluorescent analysis demonstrated that BIX01294 exposure allowed for the subsequent display of various muscle proteins within the cells. The effect of BIX01294 on the BM cell phenotype and differentiation potential corresponded to an overall decrease in methylation of histone H3 at lysine9, which is the primary target of G9a histone methyltransferase. In summary, these data suggest that BIX01294 inhibition of chromatin methylation reprograms BM cells to a cardiac-competent progenitor phenotype.

Canonical WNT signaling enhances stem cell expression in the developing heart without a corresponding inhibition of cardiogenic differentiation.

WNT signaling has been shown to influence the development of the heart. Although recent data suggested that canonical WNTs promote the emergence and expansion of cardiac progenitors in the pregastrula embryo, it has long been accepted that once gastrulation begins, canonical WNT signaling needs to be suppressed for cardiac development to proceed. Yet, this latter supposition appears to be odds with the expression of multiple canonical WNTs in the developing heart. The present study examining the effect of ectopic canonical WNT signaling on cardiogenesis in the developing frog was designed to test the hypothesis that heart formation is dependent on the inhibition of canonical WNT activity at the onset of gastrulation. Here we report that cardiac differentiation of explanted precardiac tissue from the dorsal marginal zone was not suppressed by exposure to WNT1 protein, although expression of Tbx5, Tbx20, and Nkx2.5 was selectively reduced. Pharmacological activation of WNT signaling in intact embryos using the GSK3 inhibitor SB415286 did not prevent the formation of an anatomically normal and functionally sound heart, with the only defect observed being lower levels of the cardiac transcription factor Nkx2.5. In both the explant and whole embryo studies, expression of muscle genes and proteins was unaffected by ectopic canonical WNT signaling. In contrast, canonical Wnt signaling upregulated expression of the cardiac stem cell marker c-kit and pluripotency genes Oct25 and Oct60. However, this regulatory stimulation of stem cells did not come at the expense of blocking cardiac progenitors from differentiating.

Evaluating the role of Wnt signal transduction in promoting the development of the heart.

Wnts are a family of secreted signaling proteins that are encoded by 19 distinct genes in the vertebrate genome. These molecules initiate several signal transduction pathways: the canonical Wnt, Wnt/Ca2+, and Wnt/planar cell polarity pathways. Wnt proteins have major impact on embryonic development, tumor progression, and stem cell differentiation. Wnt signal transduction also influences the formation of the heart, yet many issues concerning the involvement of Wnt regulation in initiating cardiac development remain unresolved. In this review, we will examine the published record to discern (a) what has been shown by experimental studies on the participation of Wnt signaling in cardiogenesis, and (b) what are the important questions that need to be addressed to understand the importance and function of Wnt signal transduction in facilitating the development of the heart.

Embryonic myocardium shows increased longevity as a functional tissue when cultured in the presence of a noncardiac tissue layer.

A major aim of regenerative medicine is the construction of bioengineered organs and tissue for transplantation into human patients; yet living tissue is dynamic, and thus arranging cellular and extracellular constituents into an architecture resembling normal adult organs may not be sufficient to maintain tissue stability. In this study, we used cultures of embryonic chick heart tissue as a model to explore how newly formed cardiac tissue constructs can sustain their morphological structure and functional capabilities over extended periods. During the initial days of incubation, embryonic cardiac explants will thrive as beating three-dimensional tissue aggregates. However, within the first week of culture, cardiac aggregates lose their contractile function and flatten. After 2 weeks of incubation, the cardiac cells will have spread out into a homogeneous monolayer and dedifferentiated to a noncardiac phenotype. In contrast, when the embryonic heart tissue was co-cultured with a noncardiac cell layer obtained from adult bone marrow, the cardiac aggregates maintained their contractile function, three-dimensional tissue morphology, and myocyte phenotype for a full month of incubation. The capacity of this noncardiac cell layer to sustain the phenotype and morphology of the cardiac explants was partially replicated by treatment of the heart tissue with conditioned media from bone marrow cells. These findings are discussed in regard to the importance of adjacent cell layers for facilitating organogenesis in the developing embryo and having potential utility in producing stable bioengineered tissue constructs.

Blood-borne stem cells differentiate into vascular and cardiac lineages during normal development.

Recent investigations have indicated that hematopoietic stem cells (HSCs) have the potential to differentiate into multiple non-blood cell lineages and contribute to the cellular regeneration of various tissues and multiple organs. Most studies to date on HSC potential have examined the adult, focusing on their potential to repair tissue under pathological conditions (e.g., ischemic injury, organ failure). Comparatively little is known about the physiological role of HSCs in normal tissue homeostasis in the adult, and even less of their contribution to organogenesis during prenatal development. This study reports the contribution of blood-borne cells to various organ systems of the developing embryo using a quail-chick parabiosis model. Under these conditions, the developing circulatory systems fuse between ED6-ED8, resulting in free exchange of circulating cells. Cells of quail origin, identified by quail-specific antibodies at ED15, were found in numerous organs of the parabiotic chick embryo. Circulating cells contributed to developing vasculature, where they differentiated into endothelial, smooth muscle, and adventitial tissues. In the heart, differentiation of circulating cells into cardiomyocytes was demonstrated using double immunolabeling for QCPN and sarcomeric actin or myosin. These results were confirmed by intramyocardial injection of quail bone marrow cells that were found to express markers of myocytes, coronary smooth muscle, and epicardium. Experiments using lacZ-transgenic chick embryos for a second positive cellular marker showed that fusion between chick and quail cells was a rare event. These results suggest that during development, multipotent cells are present in the embryonic circulation and home into different organs where they undergo tissue-specific differentiation. Moreover, the demonstration that blood-borne cells contribute to the development of various organs lends credence to claims that hematopoietic stem cells have utility for treating diseased or damaged tissues in the adult.

Bone marrow cells transdifferentiate to cardiomyocytes when introduced into the embryonic heart.

Since rates of cardiomyocyte generation in the embryo are much higher than within the adult, we explored whether the embryonic heart would serve as useful experimental system for examining the myocardial potential of adult stem cells. Previously, we reported that the long-term culturing of adult mouse bone marrow produced a cell population that was both highly enriched for macrophages and cardiac competent. In this study, the myocardial potential of this cell population was analyzed in greater detail using the embryonic chick heart as recipient tissue. Experiments involving the co-incubation of labeled bone marrow cells with embryonic heart tissue showed that bone marrow (BM) cells incorporated into the myocardium and immunostained for myocyte proteins. Reverse transcription-polymerase chain reaction analysis demonstrated that the heart tissue induced bone marrow cells to express the differentiated cardiomyocyte marker alpha-cardiac myosin heavy chain. The cardiomyocyte conversion of the bone marrow cells was verified by harvesting donor cells from mice that were genetically labeled with a myocardial-specific beta-galactosidase reporter. Embryonic hearts exposed to the transgenic bone marrow in culture exhibited significant numbers of beta-galactosidase-positive cells, indicating the presence of bone marrow-derived cells that had converted to a myocardial phenotype. Furthermore, when transgenic mouse BM cells were injected into living chick embryos, donor cells incorporated into the developing heart and exhibited a myocardial phenotype. Immunofluorescence analysis demonstrated that donor BM cells exhibiting myocyte markers contained only nuclei from mouse cells, indicating that differentiation and not cell fusion was the predominant mechanism for the acquisition of a myocyte phenotype. These data confirm that adult mouse bone marrow contain cells with the ability to form cardiomyocytes. In addition, the predominance of the macrophage phenotype within the donor bone marrow cell population suggests that transdifferentiation of immune response cells may play a role in cellular regeneration in the adult.

Wnt11 signaling promotes proliferation, transformation, and migration of IEC6 intestinal epithelial cells.

Wnts are morphogens with well recognized functions during embryogenesis. Aberrant Wnt signaling has been demonstrated to be important in colorectal carcinogenesis. However, the role of Wnt in regulating normal intestinal epithelial cell proliferation is not well established. Here we determine that Wnt11 is expressed throughout the mouse intestinal tract including the epithelial cells. Conditioned media from Wnt11-secreting cells stimulated proliferation and migration of IEC6 intestinal epithelial cells. Co-culture of Wnt11-secreting cells with IEC6 cells resulted in morphological transformation of the latter as evidenced by the formation of foci, a condition also accomplished by stable transfection of IEC6 with a Wnt11-expressing construct. Treatment of IEC6 cells with Wnt11 conditioned media failed to induce nuclear translocation of beta-catenin but led to increased activities of protein kinase C and Ca(2+)/calmodulin-dependent protein kinase II. Inhibition of protein kinase C resulted in a decreased ability of Wnt11 to induce foci formation in IEC6 cells. Finally, E-cadherin was redistributed in Wnt11-treated IEC6 cells, resulting in diminished E-cadherin-mediated cell-cell contact. We conclude that Wnt11 stimulates proliferation, migration, cytoskeletal rearrangement, and contact-independent growth of IEC6 cells by a beta-catenin-independent mechanism. These findings may help understand the molecular mechanisms that regulate proliferation and migration of intestinal epithelial cells.

Adult stem cells and their cardiac potential.

Adult cardiac muscle is unable to repair itself following severe disease or injury. Because of this fundamental property of the myocardium, it was long believed that the adult myocardium is a postmitotic tissue. Yet, recent studies have indicated that new cardiac myocytes are generated throughout the life span of an adult and that extracardiac cells can contribute to the renewal of individual cells within the myocardium. In addition, investigations of the phenotypic capacity of adult stem cells have suggested that their potential is not solely restricted to the differentiated cell phenotypes of the source tissue. These observations have great implications for cardiac biology, as stem cells obtained from the bone marrow and other readily accessible adult tissues may serve as a source of replacement cardiac myocytes. In this review, we describe the evidence for these new findings and discuss their implications in context of the continuing controversy over stem cell plasticity.

Hematopoietic cells from bone marrow have the potential to differentiate into cardiomyocytes in vitro.

Recent studies have indicated that hematopoietic progenitor cells (HPCs) have the capacity to form cardiomyocytes. In the present study, we further examined the cardiac competence of HPCs by asking whether these cells by themselves can be provoked to undergo cardiac differentiation. Our data indicate that in response to growth factor treatment, HPCs from avian bone marrow (BM) can undergo cardiac differentiation, as indicated by their expression of multiple cardiac transcription factors and sarcomeric proteins. Furthermore, coculture experiments with adult mouse BM cells and embryonic heart tissue confirmed that HPCs are able to both integrate into cardiac tissue and differentiate into cardiomyocytes. In an additional set of experiments, we investigated whether other hematopoietic populations might possess cardiac potential by examining whether blood cells that normally are recruited to damaged tissue might act as a source of newly generated cardiomyocytes. Remarkably, macrophages cocultured with cardiac explants also demonstrated an ability to integrate into contractile heart tissue and undergo cardiac differentiation. Thus, our data suggest that the capacity of blood cells to transdifferentiate into cardiomyocytes is not limited to classically defined hematopoietic progenitors.

Wnt11 and Wnt7a are up-regulated in association with differentiation of cardiac conduction cells in vitro and in vivo.

The heart beat is coordinated by a precisely timed sequence of action potentials propagated through cells of the conduction system. Previously, we have shown that conduction cells in the chick embryo are derived from multipotent, cardiomyogenic progenitors present in the looped, tubular heart. Moreover, analyses of heterogeneity within myocyte clones and cell birth dating have indicated that elaboration of the conduction system occurs by ongoing, localized recruitment from within this multipotent pool. In this study, we have focused on a potential role for Wnt signaling in development of the cardiac conduction system. Treatment of embryonic myocytes from chick with endothelin-1 (ET-1) has been shown to promote expression of markers of Purkinje fiber cells. By using this in vitro model, we find that Wnt11 are Wnt7a are up-regulated in association with ET-1 treatment. Moreover, in situ hybridization reveals expression, although not temporal coincidence of, Wnt11 and Wnt7a in specialized tissues in the developing heart in vivo. Specifically, whereas Wnt11 shows transient and prominent expression in central elements of the developing conduction system (e.g., the His bundle), relative increases in Wnt7a expression emerge at sites consistent with the location of peripheral conduction cells (e.g., subendocardial Purkinje fibers). The patterns of Wnt11 and Wnt7a expression observed in vitro and in the embryonic chick heart appear to be consistent with roles for these two Wnts in differentiation of cardiac conduction tissues.

Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis.

Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification, which indicates that Wnt-11 may be involved in heart development. Here we show, through loss- and gain-of-function experiments, that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of beta-catenin and involves protein kinase C and Jun amino-terminal kinase. Our results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction.